This project is to lay the groundwork for innovative experimental therapeutic approaches to treat or prevent catecholaminergic neurodegeneration. The catecholaldehyde hypothesis is based on toxicity exerted by the obligate intra-neuronal metabolite of dopamine, 3,4-dihydroxyphenylacetaldehyde (DOPAL). According to the catechoaldehyde hypothesis, a monoamine oxidase (MAO) inhibitor to decrease DOPAL production, combined with an anti-oxidant to inhibit conversion of DOPAL to DOPAL-quinone, should slow the disease process causing catecholaminergic neurodegeneration in Parkinsons disease (PD) and related disorders. (1) Catecholamine autotoxicity. Implications for pharmacology and therapeutics of Parkinson disease and related disorders: Catecholamine neurons are rare in the nervous system, and why they are vulnerable in PD and related disorders has been mysterious. According to the catecholaldehyde hypothesis for the pathogenesis of PD, long-term buildup of DOPAL contributes to the eventual death of catecholaminergic neurons. Lewy bodies, a neuropathologic hallmark of PD, contain precipitated alpha-synuclein. Bases for the tendency of alpha-synuclein to precipitate in the cytoplasm of catecholaminergic neurons have also been mysterious. DOPAL potently oligomerizes and aggregates alpha-synuclein. Therefore, DOPAL and alpha-synuclein may be nodes in a complex nexus of interacting homeostatic systems. Dysfunctions of several processes, including the double hit of decreased vesicular sequestration of cytoplasmic catecholamines and decreased aldehyde dehydrogenase activity (resulting in DOPAL accumulation) and oligomerization of alpha-synuclein could lead to conversion from the stability afforded by negative feedback regulation to the instability, degeneration, and system failure caused by induction of positive feedback loops. These dysfunctions result could from diverse combinations of genetic predispositions, environmental exposures, stress, and time. Disease modification clinical trials would provide key tests of the catecholaldehyde hypothesis.1 (2) Comparison of monoamine oxidase inhibitors in decreasing production of the autotoxic DOPAL in PC12 cells: We compared the potencies of monoamine oxidase (MAO) inhibitors in attenuating DOPAL production and examined possible secondary effects on dopamine storage, constitutive release, synthesis, and spontaneous oxidation in rat pheochromocytoma PC12 cells, which express both dopamine and DOPAL endogenously. Catechol concentrations were measured in cells and medium after incubation with the irreversible MAO-A inhibitor clorgyline, three reversible MAO-A inhibitors, or the MAO-B inhibitors selegiline or rasagiline for 180 minutes. Reversible MAO-A inhibitors were generally ineffective, whereas clorgyline (1 nM), rasagiline (500 nM), and selegiline (500 nM) decreased DOPAL levels in the cells and medium. All 3 drugs also increased dopamine and norepinephrine, decreased DOPA, and increased Cys-DA concentrations in the medium, suggesting increased vesicular uptake and constitutive release, decreased dopamine synthesis, and increased dopamine auto-oxidation. Possibly offsetting increased formation of potentially toxic oxidation products and decreased formation of DOPAL might account for the failure of large clinical trials of MAO-B inhibitors to demonstrate slowing of neurodegeneration in PD.2 (3) Hydroxytyrosol (DOPET) mitigates the increase in spontaneous oxidation of dopamine during MAO inhibition: Inhibiting MAO diverts the fate of cytoplasmic dopamine toward potentially harmful spontaneous oxidation products, indicated by increased Cys-DA levels. DOPET is an abundant anti-oxidant phenol in constituents of the Mediterranean diet. Whether DOPET alters enzymatic or spontaneous oxidation of DA has been unknown. PC12 cells were incubated with DOPET alone or with the MAO-A inhibitor clorgyline or the MAO-B inhibitors rasagiline or selegiline. Co-incubation with DOPET prevented the increases in Cys-DA levels seen with all 3 MAO inhibitors. DOPET therefore inhibits both enzymatic and spontaneous oxidation of endogenous DA and mitigates the increase in spontaneous oxidation of DA during MAO inhibition.3 (4) Plasma catechols after eating olives: Olives contain 3,4-dihydroxyphenyl compounds (catechols)especially 3,4-dihydroxyphenylethanol (DOPET)that have therapeutic potential as nutraceuticals. Whether olives ingestion affects plasma levels of free (unconjugated) catechols has been unknown. Arm venous blood was sampled before and 15, 30, 45, 60, 120, 180, and 240 minutes after 6 healthy volunteers ate 10 Kalamata olives. Catechols were assayed by alumina extraction followed by liquid chromatography with series electrochemical detection. Plasma DOPET increased to 18.5 times baseline at 30 minutes (area under the curve (AUC) 39.2 9.2 pmol-min/mL, p=0.008). 3,4-Dihydroxyphenylacetic acid (DOPAC) increased markedly (peak 37.4 times baseline, AUC 23,4904151 pmol-min/mL, p=0.002). The sum of 10 catechols increased 12-fold (p<0.0001). Eating olives produces large-magnitude increases in plasma levels of catechols, mainly DOPAC. DOPET seems to go undergo extensive hepatic metabolism to DOPAC.4 (5) N-Acetylcysteine prevents the increase in spontaneous oxidation of dopamine during monoamine oxidase inhibition in PC12 cells: The catecholaldehyde hypothesis predicts that inhibiting MAO should slow the progression of PD, by decreasing DOPAL production. MAO inhibition, however, increases spontaneous oxidation of dopamine, as indicated by Cys-DA levels, and the oxidation products may also be toxic. We examined whether N-acetylcysteine (NAC), a precursor of the anti-oxidant glutathione, attenuates the increase in Cys-DA production during MAO inhibition. PC12 cells were incubated with NAC, the MAO-B inhibitor selegiline, or both. Selegiline decreased DOPAL and increased Cys-DA levels (p<0.0001 each). Co-incubation of NAC at pharmacologically relevant concentrations (1-10 M) with selegiline (1 M) attenuated or prevented the Cys-DA response to selegiline, without interfering with the selegiline-induced decrease in DOPAL production or inhibiting tyrosine hydroxylation. NAC therefore mitigates the increase in spontaneous oxidation of dopamine during MAO inhibition.5 REFERENCES 1. Goldstein DS, Kopin IJ, Sharabi Y. Catecholamine autotoxicity. Implications for pharmacology and therapeutics of Parkinson disease and related disorders. Pharmacol Ther 2014;144:268-282. 2. Goldstein DS, Jinsmaa Y, Sullivan P, Holmes C, Kopin IJ, Sharabi Y. Comparison of monoamine oxidase inhibitors in decreasing production of the autotoxic dopamine metabolite 3,4-dihydroxyphenylacetaldehyde in PC12 cells. J Pharmacol Exp Ther 2016;356:484-493. 3. Goldstein DS, Jinsmaa Y, Sullivan P, Kopin IJ, Sharabi Y. 3,4-Dihydroxyphenylethanol (hydroxytyrosol) mitigates the increase in spontaneous oxidation of dopamine during monoamine oxidase inhibition in PC12 cells. Neurochem Res 2016;41:2173-2178. 4. Goldstein DS, Holmes C, Cherup J, Sharabi Y. Plasma catechols after eating olives. Clin Trans Sci 2017 (in press). 5. Goldstein DS, Jinsmaa Y, Sullivan P, Sharabi Y. N-Acetylcysteine prevents the increase in spontaneous oxidation of dopamine during monoamine oxidase inhibition in PC12 cells. Neurochem Res 2017 (in press).